Zeb2 Chip-Seq in Stem Cells Zeb2 DNA-Binding Sites in ES Cell

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Zeb2 Chip-Seq in Stem Cells Zeb2 DNA-Binding Sites in ES Cell bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Zeb2 ChIP-seq in stem cells Zeb2 DNA-binding sites in ES cell derived neuroprogenitor cells reveal autoregulation and align with neurodevelopmental knockout mouse and disease phenotypes. Judith C. Birkhoff1, Anne L. Korporaal1, Rutger W.W. Brouwer2, Claudia Milazzo1#, Lidia Mouratidou1, Mirjam C.G.N. van den Hout2, Wilfred F.J. van IJcken1,2, Danny Huylebroeck1,3*, $, Andrea Conidi1*, $ 1 Dept. Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands; 2 Center for Biomics-Genomics, Erasmus University Medical Center, Rotterdam 3015 CN, The Netherlands; 3 Dept. Development and Regeneration, KU Leuven, Leuven 3000, Belgium * shared senior authors # Present address: Dept. Clinical Genetics, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands $ Corresponding author: Andrea Conidi, PhD, Dept. Cell Biology, Erasmus MC, Building Ee - room Ee-1040, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands; Tel: +31-10-7043169; Fax: +31-10-7044743; E-mail: a.conidi@erasmusmc.nl 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Author contributions JB: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing AK: Collection and/or assembly of data RB: Data analysis and interpretation CM: Collection and/or assembly of data LM: Collection and/or assembly of data MvdH: Collection and/or assembly of data, data analysis and interpretation WvIJ: Data analysis and interpretation, manuscript writing DH: Conception and design, financial support, manuscript writing, final approval of manuscript AC: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript Key words Embryonic stem cells; genome-wide binding sites; Mowat-Wilson syndrome; neuroprogenitor cells; Zeb2 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Perturbation and mechanistic studies have shown that the DNA-binding transcription factor Zeb2 controls cell fate decision, differentiation and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs) Zeb2’s strong upregulation is necessary for their exit from primed pluripotency and entering neural and general differentiation. We engineered mouse ESCs to produce epitope-tagged Zeb2 from one of its two endogenous alleles. Using crosslinking ChIP-sequencing, we mapped for the first time 2,432 DNA-binding sites of Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major site maps promoter-proximal to Zeb2 itself, and its homozygous removal demonstrates that Zeb2 autoregulation is necessary to elicit proper Zeb2 effects in ESC→NPC differentiation. We then cross-referenced all Zeb2 DNA-binding sites with transcriptome data from Zeb2 perturbations in ESCs, ventral forebrain in mouse embryos, and adult neurogenesis. While the characteristics of these neurodevelopmental systems differ, we find interesting overlaps. Collectively, these new results obtained in ESC-derived NPCs significantly add to Zeb2’s role as neurodevelopmental regulator as well as the causal gene in Mowat-Wilson Syndrome. Also, Zeb2 was found to map to loci mutated in other human congenital syndromes, making variant or disturbed levels of ZEB2 a candidate modifier principle in these. Significance statement Zeb2 is needed for exit from primed pluripotency and differentiation of ESCs and being studied in various cell lineages and tissues/organs in knockouts and adult mice. Phenotyping has mainly documented transcriptomes of Zeb2-mutant affected cell types, but ChIP-seq data are still not available to document Zeb2’s direct target loci as repressor or activator of transcription. This study maps for the first time the Zeb2 genome-wide binding sites in ESC-derived neuroprogenitors, and discovered Zeb2 autoregulation. Cross-referencing these ChIP-seq data with transcriptome profiles of 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Zeb2 perturbation further explains Mowat-Wilson Syndrome defects and proposes ZEB2 as candidate modifier gene in other syndromes. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Zeb2 (also named Sip1/Zfhx1b) and Zeb1 (δEF1/Zfhx1a), the two members of the small family of Zeb transcription factors (TFs) in vertebrates, bind to two separated E2-boxes CACCT(G), sometimes CACANNT(G), on DNA via two highly conserved, separated clusters of zinc fingers [1-4]. Mutations in ZEB2 cause Mowat-Wilson Syndrome (MOWS, OMIM#235730), a rare congenital disease with intellectual disability, epilepsy/seizures and other defects [5-8]. Zeb2’s action mechanisms, partner proteins, proven or candidate target genes, and genes whose normal expression depends on intact Zeb2 levels, have been studied in various cell types for explaining specific phenotypes caused by Zeb2 perturbation. Zeb2 DNA-binding around candidate direct target genes helped to explain Zeb2 loss-of- function phenotypes in ESCs and cells of early and late embryos, and postnatal and adult mice. These genes are involved in ESC pluripotency (Nanog, Sox2), cell differentiation (Id1, Smad7), and maturation of various cell types, e.g., in embryonic cortical and adult neurogenesis (Ntf3, Sox6), as well as epithelial-to-mesenchymal transition (EMT) (Cdh1) [9-16]. In reverse, subtle mutagenesis of Zeb2 DNA-binding sites in demonstrated target genes has confirmed Zeb DNA-binding and its repressive activity (on the mesoderm TF XBra and on epithelial Cdh1) [9, 17]. Despite its critical function in the precise spatial-temporal regulation of expression of system/process-specific relevant genes during embryogenesis and postnatal development, but recently also adult tissue homeostasis and stem cell based repair, and acute and chronic disease [18, 19], ChIP- sequencing (ChIP-seq) data for Zeb2 has been obtained in very few cases only. This is the case for high-Zeb2 hepatocellular carcinoma and leukemia cell lines, or cultured cells that overproduce tag- Zeb2 from episomal vectors or the safe Rosa26 locus [15, 20-22], none of which represent normal, endogenous Zeb2 levels nor express Zeb2 with normal dynamics. Furthermore, most if not all anti- Zeb2 antibodies cross-react with Zeb1, so do not discriminate between both proteins when their presence overlaps or succeeds to one another, and they compete for the same target genes, which for the individual proteins depends on cell identity/state, extrinsic stimulation or cellular context (e.g., in somitogenesis, in melanoma) [23,24]. In mouse (m) ESCs subjected to neural differentiation (ND), Zeb2 mRNA/protein is undetectable in undifferentiated cells, whereas its strong upregulation accompanies efficient 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.06.451350; this version posted July 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. conversion of naïve ESCs into epiblast stem cell like cells (EpiLSCs), and is essential for subsequent exit from primed cells and progression to neuroprogenitor cells (NPCs) [15]. We have edited one Zeb2 allele of mESCs by inserting a Flag-V5-epitope coding sequence just before the stop codon, in-frame with the last exon (ex9 of mouse Zeb2) [25]. These Zeb2-V5 mESCs were then differentiated to NPCs, and Zeb2 binding sites determined by V5-tag Chip-Seq. We identified ~2,430 binding sites, of which ~1,950 map to protein-encoding genes. We then cross-referenced the ChIP-positive (+) protein- encoding target genes with RNA-seq data of deregulated genes in cell-type specific neurodevelopment-relevant Zeb2 perturbations [13, 25, 26]. Although we compare non-identical systems, the ESC approach still revealed a number of interesting overlaps, as well as Zeb2’s role in regulating critical targets in neurodevelopment. Taken together, we report for the first time the identification of Zeb2 genome-wide binding sites in ESC neural differentiation. Materials and Methods Tag-Zeb2 mouse ESCs gRNAs (Table 1) targeting Zeb2-ex9, and tracrRNA (Integrated DNA Technologies, IDT), were diluted to 125 ng/µl in duplex buffer (IDT). gRNAs were annealed to tracrRNA at 1:1 ratio at 95°C for 5 min and cooling the samples to room temperature (RT, 24°C). 250 ng of these annealed gRNAs were transfected in 350,000 mESCs together with 2 µg pX459-Cas9-puro vector and 1 µg ssDNA oligo of the Donor Template containing the FlagV5-tag sequence (Table 1). Transfection was done in a gelatin-coated 6-well plate using DNA:Lipofectamine2000 (ratio of 1:2). Six hours after transfection the medium was refreshed, at 24 hours the cells were Puromycin-selected (2 µg/ml). After 2 days, the remaining cells were transferred to gelatin-coated 10-cm dishes and given fresh ESC-medium (see below). Per dish 1,000; 1,500; or 2,000 cells were plated and allowed to form colonies. Medium was changed every other day. Colonies were picked, expanded and genotyped by PCR (both outer and inner primer sets were used (Table S1; Fig. S1).
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